Proton transfer reactions in proteins are fundamentally important in bioenergetics and biochemical catalysis. During FY00, we have focused on proton translocation in cytochrome c oxidase, the terminal enzyme of the respiratory chain. Cytochrome c oxidasecatalyzes the reduction of molecular oxygen to water. This is coupled to proton translocation across the mitochondrial or bacterial membrane, by which energy is conserved for the synthesis of ATP. In collaboration with Marten Wikstrom's group (University of Helsinki), we could show that oxygen reduction and proton translocation in cytochrome c oxidase can take place without a highly conserved glutamic acid. Double and triplemutant enzymes were designed based on the amino acid sequences of thermophilic relatives. Our molecular dynamics simulations show that in the mutant enzymes the proton translocating function of the glutamic acid is taken over by a tyrosine which supports a transient array of water molecules. These results provide strong evidence for water-mediated proton transfer into the active site of heme-copper oxidases; and they put important constraints on the proton-pumping mechanism and kinetics cytochrome c oxidase, in particular regarding the path of protons and the rate limiting step in oxygen reduction.